Insulators, enhancers and promoters are transcription regulatory sequences critical for the establishment of specific patterns of gene expression during cell differentiation. It is becoming increasingly evident that these sequences interact in the nuclear space over long distances to regulate the establishment of distinct transcriptional profiles characteristic of specific cellular fates. The requirement for local interactions at specific loci, for example between enhancers and promoters has been studied in detail. However, the extent and function of long-range inter- and intra-chromosomal interactions mediated by these sequences at the level of the whole genome and its role in establishing and/or maintaining specific gene expression profiles is largely unexplored. We propose to carry out experiments to understand the relationship between the establishment and maintenance of patterns of transcription during cell differentiation and reprogramming and the three-dimensional arrangement of the chromatin fiber in the nuclear space. The 3P arrangement of the chromatin during S and M phases will be analyzed by Hi-C and results will be compared to those obtained in Gl cells in order to understand the role of insulators in the control of genome architecture and function throughout the cell cycle. We will perform functional experiments to test the hypothesis that transcription and insulators collaborate in the establishment of chromosome topological domains. Point to point interactions between enhancers, promoters and insulators will be analyzed by ChlA-PET in order to understand the mechanisms by which insulators control the formation of these domains. We will establish detailed maps of intra- and inter-chromosomal interactions in the genome of human stem cells and their progenitors as they differentiate into pancreatic cells and these cells are reprogrammed into IPSCs. Results from these experiments will shed light on the mechanisms by which insulators regulate the 3P organization of the genome as it relates to different nuclear processes. This organization may serve as a fingerprint of cell identity that synthesizes the epigenetic status of the cell, and may help to identify the state of differentiated, trans-differentiated or reprogrammed cells for use in human cell therapy.